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Ribonucleosides such as Ribavirin have been used as antivirals independently and in combination with other drugs for many years. Ribavirin, 1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, has a carboxamide substituent on a 1,2,4 triazole ring. The antiviral spectrum for Ribavirin was the first broad band antiviral, which did not induce interferon. In invitro tissue culture it was demonstrated to be active against at least 16 DNA and RNA viruses. (Science, 177, 705, 1972). More recently, the L isomer of Ribavirin has shown immuno-modulatory activity similar to Ribavirin but with a better tolerability profile. Phase I clinical trials were initiated in February 2001.
Ribonucleosides also have the potential as therapeutic agents in oncology, since they target mRNA resulting in the prevention of gene translation. Additionally, there is a potential therapeutic application in the treatment of ocular diseases. TRIBOSE can be considered the next generation ribonucleoside.
A significant differentiation between TRIBOSE and Ribavirin is in the triazole ring structure. Ribavirin contains a 1,2,4 triazole ring whereas TRIBOSE contains a 1,2,3 triazole ring. Other key differences are with different substitutents on the triazole ring. A 1,2,3-triazole ribonucleoside, (TRIBOSE), has not been tested for pharmacological activity either with specific targets or in general. The present invention is such a triazole. Not only has the Ribonucleoside described never been previously synthesized or tested, the nucleoside itself is not described in the literature. Although it is prepared from known and commercially available materials and the synthesis of the key intermediate 3-Isoxazolecarboxylic acid, 5-methyl-,2-(phenylmethyl)-hydrazide has been a commercial drug for many years and marketed around the world as an antidepressant, the nucleoside prepared from it has not been tested for pharmacological activity.
The final chemical step of the process to prepare this nucleoside is also one of the claims of the present invention. It is a thermal isomerization of 5-Methyl-3-isoxazolecarboxylic acid 2-(phenylmethyl) hydrazide to form the 1,2,3 triazole as shown in FIG. 2.
In addition to potential anti-viral activity, its potential for anti-neoplastic activity, TRIBOSE 1 can be viewed as an anti-sense drug. Altering the activity of the target RNA or interference with replication on a cellular level can include several mechanisms. One may be the steric hindrance resulting from the substituants on the triazole ring. A second can be due to the hydroxy function, which because of its relationship with the hydroxy function of ribose can mimic ribose and interfere with replication. The hydroxy function on the triazole ring is also subject to phosphorylation, which can enhance the antitumor activity. The similarity of the five membered ring of the triazole and the five membered ring of ribose also has the potential to interfere with the fusion reaction between DNA and mRNA. These are but three examples of how TRIBOSE can potentially interfere on a cellular level with the replication of viruses and or tumors. A significant difference between Ribavirin and TRIBOSE is in the symmetry of TRIBOSE having a 1,2,3-triazole ring as opposed to the 1,2,4-triazole ring of Ribavirin.
By having the potential to mimic the biochemical activity of nucleic acids, TRIBOSE and its derivatives enter into a cell""s nucleic acid, namely RNA and DNA, and do not permit the continuance of nucleic acid synthesis which accounts for their effectiveness. Because of the potential toxic effects to normal cells, administration by direct infusion into the tumor has the potential for maximum effectiveness with minimal side effects.
This invention relates to the novel Ribonucleoside TRIBOSE and derivatives of TRIBOSE as depicted in FIG. 4 where R1, R2, and R3 represent alkyl, acyl, hydroxy, keto, amino, phenyl, and the like. Synthesis and evaluation of TRIBOSE analogs have the potential to obtain a target molecule, which is more effective, less toxic and better tolerated than TRIBOSE. The optimum TRIBOSE candidate would target cancerous cells while leaving normal cells unchanged.
In addition to potential increased efficacy and tolerability, a key advantage of TRIBOSE as compared to other Ribonucleosides is the relative ease of synthesis. Although it is a multi-step synthesis, it is not overly complicated, has well defined intermediates obtained in good yields and are stable. Overall, substantial quantities of TRIBOSE can be readily prepared.
FIG. 1 depicts the final chemical reaction step to produce TRIBOSE 1. Reacting a protected Ribose, having structure 3 with the triazole, having structure 2 to form the Ribonucleoside, followed by deprotection, to form structure 1 has been carried out in similar reactions such as Ribavirin.(J T Witkowski et al., J Med. Chem. 15, 1150, 1972). The reaction can be carried out either chemically or enzymatically. The present invention is the new chemical entity represented by structure 1 in FIG. 1. Claim 2 of the present invention is the preparation of compound having the structure 2 represented in FIG. 1. The preparation of2 is depicted in FIG. 2. The isomeric rearrangement of compound 4 to form compound 2 can be carried out neat or in a solvent near the melting point of compound 4. Compound 4, 3-Isoxazolecarboxylic acid, 5-methyl-,2-(phenylmethyl)-hydrazide, has been marketed as an antidepressant for many years and its preparation on a commercial scale is known, and is shown as part of FIG. 3. Once it was discovered that compound 4 could be thermally isomerized to the 1,2,3 triazole having the structure 2, several experiments were conducted under various temperatures to optimize its formation. Once produced, the material was identified through elemental analysis, mass spectroscopy, and NMR. Although not fully optimized, enough material was produced to demonstrate the practicality of producing it via a thermal isomerization.